Members of the trypsin/chymotrypsin-like (S1) serine protease family play pivotal roles in a multitude of diverse physiological processes, including digestive processes and regulatory amplification cascades through the proteolytic activation of inactive zymogen precursors. In many instances protease substrates within these cascades are themselves the inactive form, or zymogen, of a xe2x80x9cdownstreamxe2x80x9d serine protease. Well-known examples of serine protease-mediated regulation include blood coagulation, (Davie, et al (1991). Biochemistry 30:10363-70), kinin formation (Proud and Kaplan (1988). Ann Rev Immunol 6:49-83) and the complement system (Reid and Porter (1981). Ann Rev Biochemistry 50:433-464). Although these proteolytic pathways have been known for sometime, it is likely that the discovery of novel serine protease genes and their products will enhance our understanding of regulation within these existing cascades, and lead to the elucidation of entirely novel protease networks.
The S1 family of serine proteases is the largest family of peptidases (Rawlings and Barrett (1994). Methods Enzymol 244:19-61). As described above, members of this diverse family perform diverse functions including food digestion, blood coagulation and fibrinolysis, complement activation as well as other immune or inflammatory responses. It is likely that these functions in both normal physiology and during diseased states, currently under investigation by numerous laboratories, will become better understood in the near future. The discovery of novel S1 serine protease cDNAs will enhance our understanding of the complex pathways controlled by these enzymes. These functions will undoubtedly be aided by the ability to express large amounts of the active protease, which is then amenable to biochemical analyses.
In the vast majority of cases, maturation of an S1 serine protease zymogen into an active form by proteolytic cleavage, results in transformation into a protease of enhanced catalytic efficiency. Zymogenicity (Tachias and Madison (1996). J Biol Chem 271:28749-28752), the degree of enhanced catalytic efficiency, varies widely among individual members of the serine protease family. Proteolytic cleavage of the conserved amino terminus zymogen activation sequence results in an aliphatic amino acid, most frequently isoleucine (Ile-16 chymotrypsin numbering), becoming protonated and thus, positively charged. The event that accompanies zymogen activation is the creation of a rigid substrate specificity pocket generated by a salt bridge between the aliphatic amino acid and a highly conserved residue aspartic acid (Asp-194 chymotrypsin numbering) one amino acid upstream from the active-site serine (Ser-195 chymotrypsin numbering) within the catalytic domain (Huber and Bode (1978). Acc Chem Res 11:114-22).
Proteases are used in non-natural environments for various commercial purposes including laundry detergents, food processing, fabric processing and skin care products. In laundry detergents, the protease is employed to break down organic, poorly soluble compounds to more soluble forms that can be more easily dissolved in detergent and water. In this capacity the protease acts as a xe2x80x9cstain remover.xe2x80x9d Examples of food processing include tenderizing meats and producing cheese. Proteases are used in fabric processing, for example, to treat wool in order prevent fabric shrinkage. Proteases may be included in skin care products to remove scales on the skin surface that build up due to an imbalance in the rate of desquamation. Common proteases used in some of these applications are derived from prokaryotic or eukaryotic cells that are easily grown for industrial manufacture of their enzymes, for example a common species used is Bacillus as described in U.S. Pat. No. 5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serine proteases isolated from a fungus, Tritirachium album, for use in laundry detergent compositions. Unfortunately use of some proteases is limited by their potential to cause allergic reactions in sensitive individuals or by reduced efficiency when used in a non-natural environment. It is anticipated that protease proteins derived from non-human sources would be more likely to induce an immune response in a sensitive individual. Because of these limitations, there is a need for alternative proteases that are less immunogenic to sensitive individuals and/or provides efficient proteolytic activity in a non-natural environment. The advent of recombinant technology allows expression of any species"" proteins in a host suitable for industrial manufacture.
A major drawback in the expression of full-length serine protease cDNAs has been overwhelming potential for the production of inactive zymogen. These zymogen precursors often have little or no proteolytic activity and thus must be activated by either one of two methods currently available. One method relies on autoactivation (Little, et al. (1997). J Biol Chem 272:25135-25142), which may occur in homogeneous purified protease preparations, that often requires high protein concentrations, and must be rigorously evaluated on a protease specific basis. The second method uses a surrogate protease, such as trypsin, to cleave the desired serine protease. The surrogate protease must then be either inactivated (Takayama, et al. (1997). J Biol Chem 272:21582-21588) or physically removed from the desired activated protease. (Hansson, et al. (1994). J Biol Chem 269:19420-6). In both methods, the exact conditions must be established empirically and activating reactions monitored carefully, since inadequate activation or over-digestion would result in a heterogeneous population of active and inactive zymogen protein. Some investigators studying particular members of the S1 serine protease family have exploited the use of restriction proteinases on the activation of zymogens expressed in either bacterium (Wang, et al. (1995). Biol Chem 376:681-4) or mammalian cells (Yamashiro, et al. (1997). Biochim Biophys Acta 1350:11-14). In one report, the authors successfully engineered the secretion of proteolytically processed and activated murine granzyme B by taking advantage of the endogenous yeast KEX2 signal peptidase in a Pichia pastoris expression system (Phain et al. (1998). J. Biol. Chem. 273:1629-1633). U.S. Pat. No. 5,326,700 shows modification of the tissue plasminogen activator (t-PA) molecule such that the polypeptide is cleaved by the expression host cell to yield mature protein upon secretion from the cell. This example of a specific modification, while simple, suffers from the requirement that the associated protease is expressed within the host cell at such levels as to cleave the t-PA, which would be expressed in large quantities relative to other host proteins. Similarly, U.S. Pat. Nos. 5,270,178 and 5,196,322 describe modification of the protein C cleavage site such that it becomes a more efficient substrate of the protease thrombin. These examples of activating recombinant zymogens clearly have the added value to permit expression and activation of several serine proteases, however there remains unmet needs in the field. The example of Pham et al clearly limits the expression system available for use due to the nature of the signal peptide. The other examples describe enzyme specific engineered constructs that do not easily predict a generic method to which other serine proteases may be applied.
Introduction of proteolytic cleavage sites into fusion proteins is well known in the art. However, it is the present invention, for the first time, that creates a fusion protein designed for the generic activation of S1 serine proteases by the introduction of a propeptide region with a predefined, easily processed, cleavage site. Inclusion of the catalytic domain of a serine protease into the fusion gene allows the specific enzyme""s activity to be preserved without the requirement of a specific activating enzyme. Because the protein is proteolytically processed using commercially available enzymes after expression in the host cell, the fusion proteins of the present invention can be expressed in any suitable cell line, including prokaryotic, eukaryotic, yeast, and insect cell lines well known in the art.
The unmet need of a genetic method to express enzymatically active serine protease is described by the current invention that provides a nucleic acid cloning method to extract the catalytic domain from any serine protease. The extracted catalytic domain may then be manipulated to simplify purification, and then expressed in any suitable cell type including bacteria, yeasts, and eukaryotic cells. Herein we describe enzymatically active, human serine proteases herein termed, prostasin (Yu et al. (1995). J. Biol. Chem. 270:13483-9), O (Yoshida, S. et al. (1998). Biochim. Biophys. Acta 1399, 225-228), neuropsin (Yoshida, S. et al. (1998). Gene 213, 9-16), F (Inoue, M., et al (1998). Biochem. Biophys. Res. Commun. 252, 307-312.) and MH2 (Nelson et al. (1999). Proc. Natl. Acad. Sci. U. S. A. 96:3114-3119). Isolation of any one or more of these purified, enzymatically active proteases allows the protein to be used directly, for the treatment of certain diseases or as an additive in commercial products. For example, isolation of purified, enzymatically active protease O allows the protein to be used directly, for the treatment of certain skin diseases or to enhance skin pigmentation. Isolation of purified, enzymatically active protease F allows the protein to be used directly, for example, for the treatment of inflammatory disease or in reproductive development, since it is expressed in eosinophils and testis (Inoue et al. (1998). Biochem. Biophys. Res. Commun. 252:307-312) or as an additive in commercial products. Since protease MH2 is prostate specific (Nelson et al. (1999). Proc. Natl. Acad. Sci. U. S. A. 96:3114-3119), it may be used as a marker for certain grades of prostate cancer. Thus, the identification of sensitive protease MH2 substrates, which would be facilitated with an active protease MH2 preparation, may result in a more reliable diagnostic marker for prostate cancer medical evaluation. Isolation of any one of these purified, enzymatically active proteases will allow them to be used directly as therapeutic proteins, for example, for the treatment of neurological function, particularly in memory functions, as well as in dermatological diseases or pancreatic insufficiency. In addition, they may be used as an additive in commercial products. Because these proteases are derived from a human host, they are less likely to induce an allergic reaction in sensitive individuals, and therefore proteases prostasin, O, neuropsin, F and MH2 could also be useful for formulation of compositions for laundry detergents and skin care products. Alternatively, enzymatically active proteases prostasin, MH2, F, O, and neuropsin may be used to discover chemical modulators of the enzyme that may be useful for treatment of the aforementioned physiological and pathological states.
The present invention provides a series of DNA vectors allowing for the systematic expression of heterologous inactive zymogen proteases that can subsequently be proteolytically processed to generate the active enzyme product. The present invention provides a system that allows generic expression and activation of S1 protease family members in bacteria, yeasts, or eukaryotic cells.
The protein products of serine protease cDNAs generated within this particular zymogen activation system can be proteolytically activated, whereby the recombinant protein will become activated to an extent similar to its mature activated gene product counterpart from native or endogenous sources.
Enzymatically active proteases MH2, F, prostasin, O, and neuropsin or any other protease are amenable to further biochemical analyses for the identification of physiological substrates and specific modulators. Modulators identified in the chromogenic assay disclosed herein are potentially useful as therapeutic agents in the treatment of diseases associated with, but not limited to, inflammatory, reproductive, epidermal and neurological tissues. Isolation of purified, enzymatically active proteases MH2, F, prostasin, O, and neuropsin or any other protease allows the proteins to be used directly, for example, for the treatment of diseases associated with, but not limited to, inflammatory, reproductive, epidermal and neurological tissues. Purified proteases MH2, F, prostasin, O, and neuropsin or any other protease can be manufactured as a component for use in commercial products including laundry detergents, stain-removing solutions, and skin care products.